Innate immune defense sensors and signaling pathways represent the first line of defense against infection by microbial pathogens, including HIV, the causative agent of AIDS. The goals of this specific project are to help define the immune defense pathways that regulate specific stages ofthe HIV-1 replication cycle, and to contribute to the development of kinetic mathematical models that can explain, and predict, the early innate immune response to HIV-1 infection. The early steps of virus replication that will be analyzed are those leading up to virus-cell membrane fusion, reverse transcription, nuclear import, and viral DNA integration. The late steps that will be analyzed are viral gene expression, RNA splicing, translation, and virus assembly/release. These studies exploit a unique dataset that includes candidate innate immune factors that were identified either through genome-wide siRNA or cDNA screening for their effects on HIV-1 infection. There is substantial overiap between these candidate genes and those in the innate immune database, interferon-stimulated genes, and genes under positive selection. However, approximately 15% of the candidate genes have not been implicated before in innate immune responses and these are potentially new players in the innate response to HIV-1. Our studies will also evaluate the roles of HIV-1 accessory proteins Vif, Vpr, Nef, and Vpu, as candidate viral countermeasures of specific innate immune factor functions. Taken together, these studies will significantly advance the understanding of the innate immune response to HIV-1 infection at a systems-wide level and they will contribute to the development of mathematical models that can predict the nature of these innate responses, providing valuable new insights into antiviral and vaccine approaches.